Autoantibodies to the catalytic domain of v-raf murine sarcoma viral oncogene homologue B1 (BRAF) have been recently identified as a new family of autoantibodies involved in rheumatoid arthritis (RA). The objective of this study was to determine antibody responses to the catalytic domain of BRAF in RA and other autoimmune diseases. The association between RA-related clinical indices and these antibodies was also assessed.The presence of autoantibodies to the catalytic domain of BRAF (anti-BRAF) or to peptide P25  was determined in serum samples from patients with RA, primary Sjögren's syndrome (pSS), systemic lupus erythematosus (SLE), and healthy controls by using indirect enzyme-linked immunosorbent assays (ELISAs) based on the recombinant catalytic domain of BRAF or a synthesized peptide, respectively. Associations of anti-BRAF or anti-P25 with disease variables of RA patients were also evaluated. Our results show that the BRAF-specific antibodies anti-BRAF and anti-P25 are equally present in RA, pSS, and SLE patients. However, the erythrocyte sedimentation rate (ESR) used to detect inflammation was significantly different between patients with and without BRAF-specific antibodies. The anti-BRAF-positive patients were found to have prolonged disease, and active disease occurred more frequently in anti-P25-positive patients than in anti-P25-negative patients. A weak but significant correlation between anti-P25 levels and ESRs was observed .The antibody response against the catalytic domain of BRAF is not specific for RA, but the higher titers of BRAF-specific antibodies may be associated with increased inflammation in RA. Autoimmune diseases occur when the body's immune system attacks self-antigens. This induces prolonged inflammation and subsequent tissue destruction. Rheumatoid arthritis (RA), a common systemic autoimmune disease of unknown etiology, is characterized by chronically inflamed synovial joints and subsequent destruction of cartilage and bones. Despite decades of research, the pathogenesis of RA is still unresolved. One of the hallmarks of RA is the presence of a broad spectrum of autoantibodies against aberrantly expressed autoantigens. The discovery of autoantibodies to citrullinated proteins such as fibrin and vimentin in patients with RA was one of the most important findings in rheumatology research in vitro. This indicates that anti-BRAF may play a role in inflammation in RA through activation of the MAPK pathway BRAF is a serine-threonine kinase involved in the mitogen-activated protein kinase (MAPK) pathways that regulate cell survival, proliferation, differentiation, cytokine generation, and metalloproteinase production Written informed consent was not obtained because of the nature of the study design, which utilized serum samples taken after routine tests. All subjects recruited in this study were informed of the nature of the project and verbal informed consent was obtained from each patient, This was recorded by the physician who explained the study procedure. The study protocol and verbal consent document were approved by the Ethics Committee of the National Center for Clinical Laboratories, where the study was performed.The DNA segment corresponding to the catalytic domain of wild-type BRAF (amino acids 416–766) was generated by PCR using specific primers carrying restriction sites. The pEF-myc-BRAF plasmid containing full-length human BRAF cDNA, was kindly provided by Dr. Richard Marais . Enzyme-restricted PCR products were ligated into the multiple cloning sites of the pET28b expression vector by T4 DNA ligase. The desired clones were confirmed by sequencing.Escherichia coli BL-21(DE3). Further, a 6× His-tagged protein was expressed with induction by 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) for 4 h at 37°C. Bacterial pellets from a total of 1 L of culture were resuspended in 10 mL lysis buffer . After the suspension was prepared, lysozyme  was added to a final concentration of 0.2 mg/mL, followed by incubation at room temperature (RT) for 30 min. The cells were further disrupted by sonication on ice for 10 min . The homogenate was then centrifuged at 4°C for 30 min at 6000 g. The supernatant was discarded, and the inclusion bodies were collected. The collected precipitates were resuspended in 10 mL washing buffer  and incubated at RT for 20 min. The inclusion bodies were then recovered by centrifugation at 4°C for 30 min at 8000 g. The above washing step was repeated twice, the inclusion bodies were dissolved in binding buffer , and the recombinant protein was further purified by affinity chromatography on a Ni-Sepharose Fast flow (FF) column . The His-tagged protein was eluted with a linear concentration gradient of imidazole from 40 to 400 mM. The fractions containing the target protein were pooled, dialyzed to remove imidazole, and stored in the presence of 6 M guanidine-HCl at −20°C. The protein concentration was determined by a standard bicinchoninic (BCA) protein assay . To evaluate the size and purity of the recombinant protein, samples were denatured in SDS loading buffer , separated on a 10% polyacrylamide gel, and stained with Coomassie blue.The recombinant plasmid carrying the catalytic domain of wild-type BRAF (pET28b-BRAF) was transformed into Serum samples were obtained from a previously described RA cohort that fulfilled the American College of Rheumatology (ACR) criteria for RA Specific antibodies to the recombinant catalytic domain of wild-type BRAF were identified in sera by an indirect ELISA. To conduct the assay, 100 µL of the recombinant catalytic domain of BRAF (2.5 µg/mL) was incubated in an ELISA plate  at 4°C overnight. Microwells were then washed with phosphate-buffered saline  with 0.05% Tween-20 (PBST). Unbound sites were blocked by incubation with 200 µL 20% newborn calf serum (NCS) in PBS at 37°C for 1.5 h. Sera were diluted 1∶200 in blocking buffer and aliquots of 100 µL were added to the wells. Wells coated with bovine serum albumin (BSA) were prepared for each sample, to assess non-specific binding. After incubation at 37°C for 1 h, plates were washed 3 times with PBST. Subsequently, the captured antibodies were detected by a horseradish peroxidase (HRP)-conjugated goat anti-human IgG (1∶10000) (Sigma), which was diluted with 20% NCS in PBST (100 µL/well). After incubation at 37°C for 30 min, wells were washed 5 times with PBST. Color was developed by application of 100 µL of tetramethylbenzidine (Sigma) at 37°C for 20 min. The reaction was stopped by addition of 0.5 M sulfuric acid, and the optical density at 450 nm (OD450), with 620 nm as the correction wavelength, was obtained using an ELISA plate reader .450 of sample−OD450 of the non-specific binding of the sample]/[OD450 of the positive control−OD450 of the non-specific binding of the positive control])×100.Each sample was assayed in duplicate. A positive serum sample was included in each assay and used to correct for inter-assay variations. Results were expressed as arbitrary units (AU) calculated as , an indirect ELISA for quantifying IgG autoantibodies to P25 was conducted. Serum samples from RA, pSS, and SLE patients were included in the assay. Eighty-nine of 140 healthy controls were also included to evaluate the cutoff value. To efficiently coat microwells with the peptide, BSA-conjugated P25, synthesized by the Chinese Peptide Company  via the solid-phase method, was used as an antigen. The purity of the conjugate was greater than 95%. Plates were coated overnight with BSA-P25 at a concentration of 5 µg/mL. After blocking unbound sites, the serum samples were diluted 1∶100 and incubated with the plates at RT for 1 h. Wells coated with BSA were prepared for each sample to determine non-specific binding. After washing, HRP-conjugated goat anti-human IgG was added and incubated at RT for 1 h. The plate was read at an OD of 450 nm, with 620 nm as the correction wavelength, using an ELISA plate reader.Each sample was tested in duplicate. A positive serum sample was included in each assay and used to correct for inter-assay variations. Data was processed as described in the anti-BRAF ELISA procedure.t-tests. For data not distributed normally, results are expressed as the median (range); differences between groups were analyzed using the Mann-Whitney U-test and correlations were determined by computing Spearman rank correlation coefficients. Pearson's 2-tailed χ2 test or Fisher's exact test were used to compare proportions. P values<0.05 were considered statistically significant.Statistical analyses were performed using SPSS 13.0 for Windows. For normally distributed data, results are expressed as the mean and standard deviation (mean (SD)); differences between groups were assessed by The recombinant catalytic domain of wild-type BRAF was expressed from pET28b-BRAF-transformed bacteria under IPTG induction. The expressed protein was within insoluble inclusion bodies. To obtain pure antigens, a protocol for inclusion-body extraction followed by affinity chromatography was implemented. Following extraction, recombinant proteins were predominantly identified in collected precipitates, but remained contaminated with a small quantity of host proteins. For further purification, precipitates were solubilized in 6 M guanidine-HCl and purified using nickel affinity chromatography under denaturing conditions. The His-tagged recombinant proteins were eluted with a gradient of increasing imidazole concentration and were detected as a single protein band at a molecular weight of approximately 40 kD on a 10% SDS-PAGE gel . The proThe distribution of BRAF-specific antibodies in RA, pSS, SLE and healthy control patients is shown in Of the 101 RA patients, 21 (20.8%) and 19 (18.8%) were identified as positive for anti-BRAF and anti-P25, respectively. Patients with BRAF-specific antibodies had significantly higher ESRs than patients without these antibodies (p = 0.040 for anti-BRAF and p = 0.030 for anti-P25). Patients with prolonged disease had a significantly higher prevalence of anti-BRAF (18/62) than patients with recent-onset disease (2/35) (p = 0.006). Furthermore, active disease occurred more frequently in anti-P25-positive patients than in anti-P25-negative patients (p = 0.034). Comparisons of disease indicators between patients with and without BRAF-specific antibodies are shown in Autoantibodies to BRAF, in particular anti-P25 antibodies, have been recently identified as specific markers for RA. However, this suggestion is based on the evidence that anti-P25 is specifically detected in RA patients comparing with AS and PsA. In this report, we developed indirect ELISAs on the basis of the recombinant catalytic domain of BRAF or the synthesized peptide P25 and determined the prevalence of autoantibodies to BRAF in patients with RA, pSS, or SLE and in healthy controls. Associations between anti-BRAF or anti-P25 and disease variables were investigated in the RA cohort. Our results indicate that neither anti-BRAF nor anti-P25 autoantibodies are specific markers for RA. Nevertheless, the associations between anti-BRAF or anti-P25 and disease variables suggest potential involvement of these antibodies in inflammation in RA patients.Protein arrays have been used to identify the catalytic domain of BRAF as a new autoantigen involved in RA in vitro. This indicates possible involvement of BRAF autoantibodies in the inflammatory responses of RA Multiple signal transduction pathways have been carefully investigated in RA. For instance, NF-κB and MAPK pathways are attractive for intervention in light of their ability to regulate many genes involved in immune responses A limitation of the current study is the inability to collect additional information regarding ESR and other demographic data for SLE and pSS patients who participated in this research as the disease controls, which left us unable to explore the correlation between BRAF-specific antibodies and ESRs for each patient. Further evaluation of BRAF-specific antibodies in autoimmune diseases and other inflammatory diseases would strengthen the conclusions of this study.In summary, we have observed a similar prevalence of autoantibodies to the intact catalytic domain of wild-type BRAF and a peptide derived from this domain in patients with RA, pSS, and SLE. The associations of anti-BRAF and anti-P25 with disease variables of RA suggest that BRAF-specific antibodies may participate in the inflammatory responses involved in RA. Our conclusion is that anti-BRAF catalytic domain antibodies and anti-P25 antibodies are not specific markers for RA, but the higher titers of BRAF-specific antibodies may be associated with increased inflammation in RA. This finding is contradictory to that of previous studies. The results presented here contribute to our understanding of the pathogenesis of RA and provide insights into the development of potential intervention targets for repressing inflammation. Extensive studies on antibody responses to BRAF in other autoimmune diseases such as pSS and SLE might contribute to a comprehensive understanding of its role in autoimmune disorders.


Mild cognitive impairment (MCI), defined as a transitional zone between normal cognition and dementia, requires a battery of formal neuropsychological tests administered by a trained rater for its diagnosis. The objective of this study was to develop a screening tool for MCI.One hundred ninety seven cognitively normal controls (NC), one hundred sixteen patients with amnestic MCI –single domain (aMCI-sd), one hundred ninety five patients with amnestic MCI-multiple domain (aMCI-md), and two hundred twenty eight patients with mild Alzheimer’s disease (AD) were evaluated by comprehensive neuropsychological tests and by the Memory and Executive Screening (MES).Correlation analysis showed that the three indicators of the MES were significantly negatively related with age (P<0.05), yet not related with education (P>0.05). There was no ceiling or floor effect. Test completion averaged seven minutes (421.14±168.31 seconds). The receiver operating characteristics (ROC) analyses performed on the aMCI-sd group yielded 0.89 for the area under the curve (AUC)  for the MES-total score, with sensitivity of 0.795 and specificity of 0.828. There was 81% correct classification rate when the cut-off was set at less than 75. Meanwhile, the aMCI-md group yielded 0.95 for the AUC  for the MES-total score, with sensitivity of 0.87 and specificity of 0.91, and 90% correct classification rate when the cut-off was set at less than 72.The MES, minimally time-consuming, may be a valid and easily administered cognitive screening tool with high sensitivity and specificity for aMCI, with single or multiple domain impairment. In China, the elderly above sixty years of age account for 13.26% of the population, and aging and Alzheimer’s disease (AD) have been significant public health problems  [Xiao-Ming] has [two][gray][puppies], and lives at [No. 58], [ He-xi][town], [Yong-an][county]. There are a total of 10 key points, they are the words in the square brackets. Subject gets 1 point for each key point he/she answers correctly. The subject does not score a point if his/her answer is only partially correct . The subject is allowed to repeat the key points in reverse orders . Repeat the sentence two more times and write down the subject’s answer.Note: 1).Requires rater to read the sentence continuously. Do not respond to any of the subject’s questions in between. 2). After the third time recalling, inform the subject “Please remember the sentence, I will ask you to repeat it later.” 3). The subject does not need to learn the third time if he/she answered all the key points correctly in the first or second time.Q2. ”Please generate all the things you can think of that can be used or seen in the kitchen”, count down 30 seconds and write down all the subject’s answers, even if there are more than 10.Q3. Conflicting Instructions: “When I tap twice, you tap once, and when I tap once, you tap twice.” In order for the subject to understand the rule, please demonstrate: The practitioner taps once, and the subject should tap twice; the practitioner taps twice, and the subject should tap once. Every tap should be about 2 seconds. Finish the series of number below: 1-1-2-1-2-2-2-1-1-2-1-2-2-1-1. Score method: Mark the ones he/she tapped wrong, minus 1 point for each mistake. Range score is 0–10.Note: 1). The numbers should be tapped continuously and equally. Finish the task in 30 seconds. 2). Make sure the subject understands the rules completely before starting. Once you begin, tap the numbers equally and do not respond to any interference until the task is done. 3). Avoid suggesting to the subject whether to tap or not. 4). The subject only scores when his/her finger touches the table. The tap does not count if his/her finger stops half way. 5). To avoid hurting the subject’s finger while tapping, subject can also choose pounding the table instead.Q4 Short delayed recall: 41) Use thumb to touch other four fingertips in order2) Put thumb between index finger and middle finger–scissor shape3) Put wrist on same side eye(Telescope-like gesture)–same side ear(Listen-like gesture)–mouth(Drink-like gesture)4) Do a cross 5) Luria action. Instructions: Make a fist, slice down with the edge of your palm, then close your fingers and put the back of your hand flat on the table.th time).Ask the subject to once again recall the sentence that was learned before.Q6. Inhibitory Control Test (Go/No-go test): “When I tap once, you tap once, and when I tap twice, you don’t tap.” In order for the subject to understand the rule, please demonstrate: The practitioner taps once, and the subject should also tap once; the practitioner taps twice, and the subject should not tap. Start the test after the subject understands the rule. If there are errors in the process, do not remind the subject. Every tap should be about 2 seconds. Finish the series of number below: 1-2-1-2-1-1-2-2-1-1-2-1-2-1-2. [Score method] Mark the ones he/she tapped wrong, minus 1 point for each mistake. Range score is 0–10.Note: The numbers should be tapped continuously and equally. Do not respond to any of the subject’s questions in between. Finish the task in 30 seconds.Q7 Long delayed recall: , and cerebral spinal fluid biomarkers. Additionally, there are some cognitive domains that the MES could not measure. Whether some atypical ADs, such as those manifesting of language deficit or visual spatial impairment, will miss being diagnosed, is worth further investigation.MES: Memory and executive screening; MMSE: Mini-mental status examination; CMMSE: Mini-mental status examination, Chinese version; MCI: Mild cognitive impairment; AD: Alzheimer’s disease; NC: Normal control; aMCI-sd: Amnestic mild cognitive impairment - single domain; aMCI-md: Amnestic mild cognitive impairment - multiple domain; naMCI: Non-amnestic mild cognitive impairment; AUC: Area under curve.The authors declare that they have no competing interests.GQH participated in the design of the study, data collection and drafted the manuscript. ZB performed the statistical analyses and critically reviewed the manuscript. ZQH carried out the neurological evaluation and clinical diagnosis and critically reviewed the manuscript. WB critically reviewed the manuscript and helped to perform data collection. HZ participated in the design and coordination of the study and contributed to its final version. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/12/119/prepub


E. coli (STEC) as well as E. coli O157:H7 were array-printed into microtiter plates, and serial dilutions of the bacteria were added and subsequently detected. Though antibody specificity was not sufficient for the development of an STEC serotyping method, the STEC antibody sets performed reasonably well exhibiting that specificity increased at lower capture antibody concentrations or, conversely, at lower bacterial target concentrations. The favorable results indicated that with sufficiently selective and ideally concentrated sets of biorecognition elements , this high-throughput platform can be used to rapidly type microbial isolates derived from food samples within ca. 80 min of total assay time. It can also potentially be used to detect the pathogens from food enrichments and at least serve as a platform for testing antibodies.Many rapid methods have been developed for screening foods for the presence of pathogenic microorganisms. Rapid methods that have the additional ability to identify microorganisms via multiplexed immunological recognition have the potential for classification or typing of microbial contaminants thus facilitating epidemiological investigations that aim to identify outbreaks and trace back the contamination to its source. This manuscript introduces a novel, high throughput typing platform that employs microarrayed multiwell plate substrates and laser-induced fluorescence of the nucleic acid intercalating dye/stain SYBR Gold for detection of antibody-captured bacteria. The aim of this study was to use this platform for comparison of different sets of antibodies raised against the same pathogens as well as demonstrate its potential effectiveness for serotyping. To that end, two sets of antibodies raised against each of the “Big Six” non-O157 Shiga toxin-producing  Centers for Disease Control and Prevention estimates that 31 major foodborne pathogens account for approximately 9.4 million illnesses; 56,000 hospitalizations; and 1,350 deaths per year in the United States alone –5. In adE. coli (STEC), E. coli O157:H7 as well as the “Big Six” non-O157 STEC, captured by antibodies and detected via labeling with a fluorescent, DNA intercalating stain. Though similar to a notable single tube-based microarray O-antigen typing assay for E. coli that employed a universal anti-LPS core antibody labeling approach [Detection microarrays, employing biorecognition elements that include nucleic acid probes or antibodies, have been proven to be advantageous as rapid methods for the high-throughput, multiplexed detection of foodborne bacterial pathogens and toxins –9. In thapproach , this ty2.2.1.® 384-well reaction plates  from PE Biosystems  which served as microarray “source” plates and antibodies were printed into black-walled, clear/transparent and flat-bottomed, polystyrene 96-multiwell microtiter plates with high binding (FLUOTRAC 600) surfaces from Greiner Bio-One North America Inc.  which served as “destination” plates. Antibodies to E. coli were obtained from Kirkegaard & Perry Laboratories, Inc.  and the Pennsylvania State University E. coli Reference Center . Anti-Shiga toxin-1 (Stx-1) antibody  was labeled with Alexa Fluor 555  according to kit instructions and used as a microarray fluorescent marker. E. coli O157:H7 strain B1409 was from Centers for Disease Control and Prevention , other bacterial strains were obtained from in-house stocks. Luria-Bertani broth was from Becton Dickinson . SYBR Gold was obtained from Invitrogen. Any chemicals not mentioned were at least of reagent grade.Reagents used in this research were: phosphate-buffered saline  tablets, glycerol, Tween 20, Tris-buffered saline , and bovine serum albumin  from Sigma . Plates used were MicroAmp2.2.Antibody solutions were printed into 96-well microplate wells using a Gene Machine Omnigrid Accent from Bucher  that held a single, SMP3 printing pin . Fluorescent scans of the microarrayed-microtiter plates were acquired with an LS400 laser scanner from Tecan . Centrifugation of microtiter plates was conducted in an Eppendorf model 5810R refrigerated centrifuge outfitted with an A-4-62 swinging bucket rotor . UV-Vis spectrophotometric measurements were made with a Cary 50 UV-Vis scanning spectrophotometer . A Petroff-Hausser counting chamber from Thomas Scientific  was used to enumerate bacterial cells.2.3.et al. [Individual colonies of bacteria were inoculated into 25 mL of modified Luria-Bertani broth. This was incubated at 37 °C for 18 h with shaking at 160 rpm. Serial dilutions of cultures were enumerated in quadruplicate with a Petroff-Hausser counting chamber as described by Gehring, et al. .2.4.E. coli capture antibodies were reconstituted in 50% glycerol to 1 mg/mL and diluted to various concentrations in PBS containing 5% glycerol for array printing. (The relatively high concentration of glycerol was maintained in order to prevent evaporation of the droplets and maintain a hydrated state for the capture antibodies [The non-biotinylated anti-tibodies .)2, with an SMP3 (100 μm spot diameter) pin, which delivered a volume of approx. 0.7 nL per contact stroke. The pins were manually sonicated for 5 min in distilled H2O after each daily printing routine. Columns of 8 spots per each antibody were printed with a spot separation, from edge-to-edge, of 150 μm in both “X-axis” and “Y-axis” directions. After printing, all wells were visually examined to ensure that spots were uniformly printed. Upon completion of printing, the spotted destination plates sat at RT for 1 h prior to use.Approximately 25 μL of thoroughly-mixed capture antibody solution was pipetted into separate wells of MicroAmp source plates on the microarray printer . In order to remove any air bubbles, the plates were centrifuged at 1,000 rpm (200 × g) for 2 min immediately prior to printing. Array printing was performed using the following settings: preprints/blots = 20; contact time = 0; dip and print velocity = 2 cm/s; dip and print acceleration = 10 cm/s2.5.ca. 3200 RCF) to promote analyte capture. The wells were washed twice with PBST and excess liquid was removed as above. Next, 50 μL SYBR Gold reporter solution was added to each well that was subjected to static incubation for 1 h at RT. Wells were washed twice with PBST, excess liquid was removed, the bottom of the plate was wiped clean with an ethanol-soaked tissue, the bottom was spray-dried with canned air, and then the plate was inverted and scanned  on the array scanner using single channel mode. During reporter incubation and prior to measurement, LS400 scanner lasers were turned on to warm up and stabilize for 30 min. Typical LS400 instrument scanning parameters, set and controlled via the Array-Pro Analyzer software (ver. 4.5.1.73) interface included: autofocusing in well mode, PMT gain that ranged from 100–150, 20 μm resolution, small pinhole setting, and optimization of integration time = 1.The procedure for conducting a fluorescent immunoassay  in the m3.i.e., captured) by the printed antisera (data not shown). A plausible explanation for this observation was that the antisera contained numerous globular proteins, including non-specific immunoglobulins, that either blocked the binding of anti-STEC antibodies or masked their effectiveness. Therefore, subsequent testing was conducted with immunoglobulin G (IgG) purified from the antisera using Protein A [Initial tests were conducted with Pennsylvania State University (PSU) antisera raised against each of the Big Six STECs. However, none of the bacteria tested were detected , but also based upon observations that purified PSU IgGs, diluted over 3 orders of magnitude in concentration, did not elicit any discernible difference in response at constant levels of bacterial cell exposure.Since this work focuses on proof-of-concept for a novel typing array, the data was compiled and presented in a semi-quantitative manner. This was not only justified by qualitative observation being sufficient for determining positive versus 1:100 to 1:1,000 at high dilution levels) and virtually instantaneous adsorption of the antibodies onto inexpensive polystyrene substrates . Unbound sites were blocked with BSA to prevent non-specific adsorption of bacterial cells. Serially diluted samples of live bacterial cells were added to the wells and the entire multiwell plate was centrifuged for 5 min to ensure contact of the cells to the capture antibodies. After washing, the cells were labeled with the nucleic acid stain, SYBR Gold, the wells were washed again, and the plate was subjected to laser-induced fluorescence in a microarray scanner.i.e., those with lower dissociation constants) are outcompeted by more tightly binding, specific IgGs. Note, these images were not normalized, so though fluorescence intensity appeared to increase from E. coli O26 to anti-O145 antibody, quantitative analysis revealed that there was no significant change in net response unlike the significant decrease in reaction for that serotype with anti-O103. Therefore, the results indicate a higher affinity to anti-O145 than anti-O103 for E. coli O26.Antibody-mediated capture of bacterial cells was observed in scanned images that revealed spots of fluorescence. Typical examples of these images are represented in Conversely, antibody array specificity also improved when bacterial target was relatively diluted as exhibited in E. coli O45. For example, the absence of spots in column 8 of This observation similarly indicates that either the reaction of weakly binding antibodies was rate limited with decreasing bacterial concentration or that the antibody fractions with the greatest specificity were at the highest concentrations in each anti-STEC IgG pool and therefore their binding was only observed at lower concentrations of bacterial target. Note, because of poor printing of capture antibody, the aberrant spots in versus weak fluorescence response. Select, closely related bacteria  were demonstrated to elicit false positive responses for both sets of antibodies. Most unexpected was that one strain of E. coli K-12 (ATCC 24425) heavily cross-reacted with both antibody sets. K-12 strains are known as “non-decorated” or “rough” strains that lack O-antigenic polysaccharide side chains in their lipopolysaccharide (LPS) coatings. (The LPS of these rough mutants is made up of only the Lipid A molecule and core oligosaccharides.) It may be that all anti-E. coli antibody sets contained antibodies that bind to the LPS core and that the core is accessible to the anti-core antibodies in the rough mutants. However, the (Shigatoxigenic) E. coli (used in this study) contained “decorated”  LPS that may sterically hinder and thus mask the binding of the anti-core antibodies to the LPS core. Additional non-pathogenic E. coli strains were tested to determine if the cross-reactivity was due to binding of the anti-STEC polyclonal IgGs to the K-12 LPS core.E. coli Crooks strain, another rough strain that contains a different LPS core structure (R1 core) [E. coli K-12 is not due to binding anti-STEC IgGs to either the K-12 type or R1 core, but rather to some epitope unique to the ATCC 29425 strain of K-12. The nature of the E. coli K-12 cross-reacting epitope(s) remains to be determined.The R1 core) , and two4.E. coli O157:H7 and Big Six STECs in a high-throughput, inexpensive, and rapid (ca. 80 min) assay. With improved biorecognition elements  this procedure may be used to rapidly screen and type large numbers of food samples for STEC and additional pathogenic bacteria.A multiwell, multiplex antibody microarray platform was developed that may find application in the detection and typing of bacterial cells, as well as antibody testing. The array platform was demonstrated with the simultaneous typing of


Background. Xenon seems to be an ideal anesthetic drug. To explore if next to the antagonism at the NMDA-receptor other molecular targets are involved, we tested the xenon requirement in short sleeping Drosophila shaker mutants and in na[har38]. Methods. The Drosophila melanogaster strains wildtype Canton-S, na[har38], sh102 and shmns, were raised and sleep was measured. Based on the response of the flies at different xenon concentrations, logEC50 values were calculated. Results. The logEC50-values for WT Canton-S were 1.671 , for shmns 1.711 . The logEC50-value for sh102 was 1.594 . The logEC-value of na[har38] was 2.076 . P values for all shaker mutants were P > 0.05, while na[har38] was found to be hyposensitive compared to wildtype (P < 0.05). Conclusions. The xenon requirement in Drosophila melanogaster is not influenced by a single gene mutation at the shaker locus, whereas a reduced expression of a nonselective cation channel leads to an increased xenon requirement. This supports the thesis that xenon mediates its effects not only via an antagonism at the NMDA-receptor. N-Methyl-D-Aspartate (NMDA) receptor, which is one of the glutamate-activated ion channels. The NMDA receptor is linked with synaptic functions like memory, pain and learning  , which exhibits a reduced expression of the nonselective cation neuronal channel NALCN , and wild-type Canton-S. To remove modifiers, stocks were consequently outcrossed for at least five rounds to Canton-S background as described before  (n = 32) slept 998 ± 29 minutes, so the daily sleep amount was comparable to the wildtype, as it was described before.The daily sleep amount in The anesthetic requirement of xenon was calculated from the response of the flies at different concentrations of xenon (20–80%). n = 238; P versus sh102> 0.05), for shmns 1.711 . The logEC50 value for sh102 was 1.594 . So there was no statistically significant difference in the xenon requirement of both shaker stocks compared to the wildtype. The logEC value of na[har38] was 2.076 . So na[har38] was found to be hyposensitive to xenon compared to all other tested strains. Fitted curves are shown in na[har38] flies. The logEC50-values for WT Canton-S were 1.671 (1.601–1.742 95%-confidence intervall; Caenorhabditis elegans, a non-NMDA-receptor was required for effects of xenon anesthesia  strain. These flies have reduced expression of the nonselective neuronal ion channel NALCN, which is permeably for sodium-, potassium-, and calcium ions, and which contributes to resting sodium permeability [na[har38] fruit flies used in this study, a reduced expression of the NALCN ion channel provoked altered sensitivities to halothane, isoflurane, and sevoflurane   , 22. Drosophila melanogaster, whereas a mutation in the nonselective ion channel NALCN actually has. This leads to the assumption that the shaker potassium channel is not a molecular target for xenon anesthesia.Our study shows that a single gene mutation in a voltage-gated potassium channel has no effect on the xenon requirement in


The tumorigenic ability of the 5-Aza-CdR-treated group was lower compared with that of the control group. In conclusion, the methylation state of the WWOX gene in HO-8910 cells may be reversed using 5-Aza-CdR, which may also inhibit the growth of these cells.The aim of this study was to explore the effects of 5-Aza-2′-deoxycytidine (5-Aza-CdR), a DNA methylation inhibitor, on the methylation state and function of the WWOX gene in the HO-8910 ovarian cancer cell line. The HO-8910 cells were divided into two groups, a control group and a 5-Aza-CdR-treated group. The methylation state of the WWOX gene was evaluated using a methylation-specific PCR assay. The effect of 5-Aza-CdR on the HO-8910 cells was analyzed using MTT and cell invasion assays, as well as flow cytometry. The animal models were established by intraperitoneal transplantation of the cells into nude mice. Following treatment with 5-Aza-CdR, a demethylation state was detected in the HO-8910 cells. WWOX protein expression was significantly higher in the 5-Aza-CdR-treated group compared with that in the control group. The cell growth rate at each tested time point and the number of invasive cells were lower in the 5-Aza-CdR-treated group compared with that in the control group. Flow cytometry revealed that 67.13% of the cells were arrested at the G Fetal bovine serum was purchased from Hangzhou Sijiqing Biology Engineering Materials Co., Ltd. . 5-Aza-2′-deoxycytidine (5-Aza-CdR) and MTT were produced by Sigma  and the Wizard DNA Clean-up System kits were obtained from Promega . The Taq DNA polymerases systems were obtained from Qiagen . The oligonucleotide primer was synthesized by Shanghai Shenggong Biological Engineering Co., Ltd. . The Transwell chamber and WWOX primary antibody were provided by Chemicon . BALB/c female nude mice, aged 4–6 weeks, were supplied by the Shanghai Laboratory Animal Center, Chinese Academy of Sciences . This study was approved by the ethics committee of Xuzhou Medical College .2 at 37°C in an airtight incubator. Logarithm vegetal period cells were added to the fluid, which included 5.0 μmol/l 5-Aza-CdR, to culture for 24 h. The solution was then replaced by fresh culture fluid containing the same concentration of 5-Aza-CdR. Subsequent to being cultured for 3 days, the solution was replaced by a fresh culture medium that did not contain the drug. The cells were allowed to incubate and the experiment was performed 5 days later.The HO-8910 cell line was maintained in RPMI-1640 medium (including 70 U/ml penicillin and 70 μg/ml streptomycin) supplemented with 10% fetal bovine serum. The cells were subcultured in a humidified atmosphere of 5% COThe cells were divided into two groups. Protease K-phenol extraction was used to extract the total DNA, and an ultraviolet spectrophotometer was used to determine the quantity and purity of the DNA. Agarose gel electrophoresis was performed to determine the DNA integrity. The ultraviolet spectrophotometer quantitatively adjusted the final concentration of DNA to 0.1 g/l, and the DNA was stored at −20°C. Subsequently, DNA modification, purification and PCR were conducted. The purification steps of the experiment were performed in accordance with the Wizard DNA Clean-up System kit instructions (Promega). PCR was performed using a Qiagen reaction system. The 20-μl reaction system consisted of 2 μl 10X PCR buffer , 0.4 μl dNTP, 0.4 μl upstream and downstream primers, 0.1 μl DNA Taq enzyme, 15.7 μl de-ionized water and 1 μl template. The PCR and MSP amplification conditions were 15 min at 95°C; 40 sec at 95°C, 40 sec at 60°C and 40 sec at 70°C, for 35 cycles; and 10 min at 72°C. An agarose gel electrophoresis was performed and a gel imaging system was used for the scanning analysis. The primer sequences for the methylated WWOX gene were forward, 5′-TATGGGCGTCGTTTTTTTAGTT-3′ and reverse, 5′-CAATCTCCGCAATATCGCGACA-3′. The sequences of the unmethylated primers were forward, 5′-TATGGGTGTTGTTTTTTTAHTT-3′ and reverse, 5′-CAATCTCCACAATATCACAACA-3′. The product size was 347 bp.The two groups of cells that were in the growth period were placed in 200 μl cell lysis solution  for cracking on ice, and the bicinchoninic acid (BCA) method was used to detect the protein concentration. In order to transfer the proteins to a nitrocellulose membrane, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. The membranes were incubated with with 5% skimmed milk powder for 60 min, prior to the addition of 1:1,000 anti-WWOX antibody (rabbit anti-human) and incubation overnight at 4°C. The cells were reacted with horseradish peroxidase-labeled sheep anti-rabbit antibody as a secondary antibody . This reaction took place at room temperature and was allowed to continue for 2 h. Subsequent to adding enhanced chemiluminescence (ECL) agent , the mixtures were placed in an anechoic chamber for exposure imaging with highly sensitive X-ray film. The gray value of the target and β-actin protein bands were measured and the ratio of the target band to β-actin indicated the relative expression level of the target protein.4/well and cultured for 1–6 days. Following this, 20 μl MTT working fluid was added to each well and incubated in CO2 at 37°C for 4 h. Dimethyl sulfoxide was added to terminate the reaction and the absorbance value was detected at 490 nm. Cellular growth curves were constructed based on these results.Two groups of HO-8910 cells were obtained in the growth period, and then plated into 96-well plates at a density of 1.5×10in vitro, an invasion membrane synthesized from matrix adhesive  was placed between the upper and lower areas of the Transwell chamber. A single HO-8910 cell suspension was vaccinated on the invasion membrane at 200 μl per hole, containing ~105 cells, and cultured at 37°C in 5% CO2 for 12 h. The cells and matrix adhesive were wiped on the membrane, which was fixed and stained with H&E. The number of transmembrane cells was calculated using a microscope. Each group of cells had three duplicate wells and the experiment was repeated three times.Using a Transwell chamber cell invasion assay The cells that were in the growth period were divided into two groups and digested using 0.25% trypsin. Physiological saline was used for conventional washing. The cells were centrifuged and fixed in 70% ethanol overnight prior to the addition of 100 μl phosphate buffer liquid to disperse them into single cell suspensions. The cells were analyzed using flow cytometry.A total of 20 BALB/c 4- to 6-week-old female nude mice were randomly divided into two groups, a control group and a 5-Aza-CdR-treated group, each containing 10 mice. They were maintained at a constant temperature (25–27°C) and humidity (45–50%), with fresh air and high dust-removing sterilizing and pathogen-free conditions. The mice were allowed free access to food and sterilized water.7/ml. The cells were inoculated into the abdominal cavities of the nude mice at a volume of 0.5 ml each. The nude mice were observed and the date of death was recorded. A necropsy was performed on the abdominal cavity immediately following death. The volume of abdominal fluid was calculated, and the number, weight and maximum diameter of the tumors and the abdominal viscera metastasis were observed.The two groups of cells in the growth period were digested to form single cell suspensions with a density of 4×10The tumor tissues from the nude mice in the 5-Aza-CdR-treated and control groups were obtained and weighed. The tumors were then ground with lysis buffer to extract the total protein. These were quantified using the BCA method. SDS-PAGE was performed using a 30-μg protein sample from each group to transfer the proteins to a nitrocellulose membrane. The membranes were incubated with 5% skimmed milk powder for 60 min at room temperature, and then 1:1,000 anti-WWOX antibody (rabbit anti-human) was added and the membranes were incubated overnight at 4°C. The mixture was then allowed to react with horseradish peroxidase-labeled sheep anti-rabbit antibody as a secondary antibody . This reaction took place at room temperature for 2 h. Subsequent to adding ECL agent, the mixture was placed in the anechoic chamber for exposure imaging with a highly sensitive X-ray film.All data are expressed as the group mean ± standard deviation, and were processed using SPSS 13.0 statistical software . The statistical differences between the groups of data were assessed using the Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.The WWOX gene was in a state of methylation in the HO-8910 ovarian cancer cell line. Unmethylated and partially methylated samples were identified following treatment with 5-Aza-CdR .Western blot analysis revealed that the expression level of the WWOX protein in the 5-Aza-CdR-treated group was 0.71±0.023, whereas that of the control group was 0.13±0.012. The difference was identified to be statistically significant P<0.05; .The MTT assay showed that subsequent to being cultured for 1–6 days, the absorbance values of cells in the 5-Aza-CdR-treated group were 0.312±0.013, 0.423±0.015, 0.488±0.022, 0.734±0.031, 0.791±0.019 and 0.814±0.018. The growth rate at each time point was significantly lower than that of the control group at the corresponding time P<0.05; .in vitro invasion assay showed that the number of transmembrane cells in the 5-Aza-CdR-treated group was 92.2±4.7, while 172.1±5.2 transmembrane cells were observed in the control group. This difference was identified to be statistically significant .The 0/G1 phase, and that the number of cells in the S phase was significantly lower than that in the control group . Each of the nude mice in the control group contained bloody ascites in the abdominal cavity, while two nude mice in the 5-Aza-CdR-treated group showed no significant bloody ascites. The two groups of nude mice had tumor nodules of various sizes scattered in numerous parts of the peritoneum, epiploon, diaphragm, bowel and the surface of the mesentery. Metastasis was observed in the liver, but not in the heart, kidneys, lungs or uterine appendages in the two groups. A comparison between the control and 5-Aza-CdR-treated group revealed significant differences in the survival time, number of metastases, metastatic foci weight, maximum tumor diameter and abdominal volume P<0.05; .Western blot analysis showed the expression level of WWOX protein in the tumor issue of the nude mice in the experimental group to be 0.65±0.031, while that of the control group was 0.25±0.047. The difference was statistically significant P<0.05; .DNA methylation is the only known natural chemical modification of DNA in mammals. It is one of the main and most studied factors in mammalian epigenetics. In DNA methylation, S-adenosyl-L-methionine supplies the methyl group, which is transferred to the fifth carbon atom of cytosine to generate 5-methylcytosine under the action of the methyltransferase. This may affect gene expression without causing any changes to the gene sequence. The site of DNA methylation is usually located on the CpG island in the promoter region of a gene . DNA hypEpigenetic changes are different from genetic alterations in several ways. One of these variations is that epigenetic changes are able to be reversed. Therefore, the normal regulation of cells is restored by recovering the expression of unmutated genes to achieve the purpose of the treatment. Normal human cell genes are not controlled by CpG island methylation. Therefore, the inhibition of methylation does not affect the expression of genes in normal cells. Genes in static states due to methylation are sensitive to DNA methylation inhibitors and to the abnormal expression of corresponding HLA molecules, allowing gene therapy to recover the activity –16. At pIn vitro invasion ability was a significant characteristic that was used to measure tumor metastatic potential. A chamber invasion in vitro experiment showed that the ability of the HO-8910 cells that were treated with 5-Aza-CdR to invade other tissues decreased markedly, suggesting that the invasion and cancer cell movement abilities were reduced. Flow cytometry showed that the HO-8910 cells were arrested in the S phase following treatment with 5-Aza-CdR. The number of cells in the G2/M phases decreased, indicating that the antitumor effect of the WWOX gene may work by affecting the cell cycle following demethylation. In the present study, the HO-8910 cells that were treated with 5-Aza-CdR were also inoculated into the abdominal cavity of nude mice, and the effects of WWOX demethylation on the behavior of the HO-8910 cells were studied further. It was identified that the growth rate of the demethylated HO-8910 cells in the nude mice was significantly slower than that of the control group. Tumorigenicity decreased significantly and the expression levels of the WWOX gene increased in the experimental group. The WWOX gene was observed to be associated with the occurrence and development of ovarian cancer; normal expression of the WWOX gene suppresses the occurrence of ovarian cancer.The present study revealed that the WWOX gene was in a state of hypermethylation in the HO-8910 ovarian cancer cell line. The cells were in a demethylated state following treatment with 5-Aza-CdR. Due to demethylation, WWOX gene expression increased. A cell growth curve experiment showed a decrease in the growth rate of the HO-8910 cells that were treated with 5-Aza-CdR, suggesting that 5-Aza-CdR may inactivate the WWOX gene through aberrant recovery of methylation. The WWOX gene tumor suppressor function was recovered, which inhibited further growth of the HO-8910 cells. in vivo and in vitro experiments revealed that aberrant methylation of the WWOX gene was the main reason for the reduction in gene transcription and expression, which may be closely associated with the occurrence and development of ovarian cancer. These results also indicated that the tumor suppressor activity was recovered by demethylation of the WWOX gene, which may provide new experimental evidence for the diagnosis and treatment of ovarian cancer.In the present study, the


Objectives. To evaluate lipid profile changes after anti-TNF therapy in patients with psoriatic arthritis (PsA). Methods. Fifteen PsA patients  under infliximab were included. None had dyslipoproteinemia or previous statin use. Total cholesterol (TC) and its fractions, inflammatory markers, and prednisone use were evaluated. Results. The comparisons of lipid levels between baseline and after three months (3M) of anti-TNF therapy showed that there was a significant increase in mean triglycerides  and VLDL-c  levels. In contrast, there were no differences in the mean TC (P = 0.28), LDL-c (P = 0.42), and HDL-c (P = 0.26) levels. Analysis of the frequencies of each lipid alteration at baseline and at 3M were alike (P > 0.05). Positive correlations were found between VLDL-c and CRP  and between triglycerides and CRP  levels at 3M. ESR reduction was observed after 3M (P = 0.04). Mean prednisone dose remained stable at beginning and at 3M (P = 0.37). Conclusion. This study demonstrated that anti-TNF may increase TG and VLDL-c levels in PsA patients after three months. Psoriatic arthritis (PsA) is a serious chronic inflammatory arthropathy of unknown cause that is associated with cutaneous psoriatic lesions . It is aα-inhibitors, where TNF-α is a cytokine that performs an important role in inflammatory diseases, which have shown satisfactory results for both joint and skin diseases [The treatment of PsA is conducted through the use of nonhormonal anti-inflammatory (NSAID) agents that are utilized in routine treatment and provide evident improvements in joint pain . Like codiseases , 10. Advdiseases , 12. Howdiseases , 14.Due to the paucity of studies, as well as the contradictory results in previous findings, the aim of this study was to evaluate lipid profile changes pre- and post-anti-TNF therapy treatment of PsA patients.This prospective study included 15 patients presenting with PsA . A venous blood sample was collected at fast for laboratory analysis before and after three months of anti-TNF treatment.This study was approved by the Research Ethic Committee of our University Hospital and an informed consent form was obtained from all participants.Total cholesterol (TC) and triglycerides (TGs) in the serum samples were measured enzymatically  with an RA 1000 Analyzer (Technicon Instruments Corp) , 17. TheThe C-reactive protein dosage (CRP)  was assessed by nephelometry. The erythrocyte sedimentation rate (ESR) was obtained using the modified Westergreen technique.P < 0.05).The results were presented in either means and standard deviations or percentages. Statistical analysis was performed using the GraphPad InStat version 2.00 program and Microsoft Excel, and Student's t-test to and the Fisher's exact test were used to compare the continuous and categorical results, respectively. In all the statistical tests, the level of significance was set at 5% (n = 15) or etanercept (n = 1). From a total of 26 PsA patients, 11 were excluded due to previous history of dyslipidemia or drugs use that interfere with lipid levels and one due to etanercept use. The mean age of the 15 patients included in this study was 41.9 ± 9.3 years, and 53% were male and 93% were Caucasians. The average duration of the disease was 14.6 ± 7.9 years. The patients included in this study were using either infliximab (P = 0.038). A trend to lower CRP was observed in these two moments . Regarding prednisone use, the dose was low and stable throughout the study, with a variation in the mean of equivalent doses between the beginning of the study and after three months (P = 0.24) (Twelve (80%) patients experienced improvement in skin and/or arthritis at 3 months. Analysis of inflammatory markers revealed that there was a significant reduction in ESR values between preadministration of anti-TNF and after three months  . P = 0.30), LDL-c (P = 0.39) and HDL-c (P = 0.26) levels did not showed significant differences in comparison to the values at the baseline and at three months after treatment, respectively. In relation to the TG levels  and VLDL-c levels , there was a significant increase in the levels of these lipids. The frequencies of dyslipidemia at baseline and after three months were alike (P > 0.05) , as well as between TG and CRP levels  at 3 months.The total cholesterol ( > 0.05) . TC/HDL-P = 0.32).Glucose levels did not alter significantly in the period studied  patients also presented contradictory results. In a study by Popa et al., they sought to evaluate whether anti-TNF treatment modified the cardiovascular risk in 33 patients with RA. They assessed the lipid profiles of patients before and at two weeks after the first adalimumab dose. They stated that the treatment reduced inflammation and improved the cardiovascular profile of these patients by improving their lipid profiles (HDL-c increase and LDL-c decrease) . AnotherIn a more recent study, Popa et al. investigated the effects of anti-TNF treatment on the lipid profiles of 55 RA patients after one year of treatment. They could demonstrate that the treatment modified the plasma concentrations in the lipoproteins of these patients. There was an increase in HDL-c concentrations and no changes in the atherogenic index during the first two weeks of treatment. Nevertheless, the beneficial effects were not sustained over time; there were eventually TC and LDL-c increases even though the HDL-c levels did not change for twelve months. This finding suggested a worsening of the lipid profiles and an increase in the atherogenic index of these patients, which could increase their cardiovascular risk .α promotes expression of adhesion molecules in endothelial cells, which initiates an inflammatory cascade in the vessel wall [α directly interferes with the metabolic routes of TGs and cholesterol [α administration results in an elevation of TG levels and a reduction of HDL-c levels [TNF-sel wall , 24. Addlesterol , 26. TNFc levels .Hypertriglyceridemia is a well-known factor of cardiovascular risk: high TG levels predict the subsequent occurrence of coronary artery disease . The impIt is clinically evident that PsA patients without cardiovascular risk factors or cardiovascular disease have an elevated prevalence for macrovascular disease, which is represented by an increased carotid intima-media thickness . This thIn summary, this study was able to demonstrate that patients with PsA who received anti-TNF therapy showed increased TG and VLDL levels after a three-month period. Larger prospective long-term studies are necessary in order to assess the impact of these lipid alterations in the cardiovascular disease in that group of patients.The author declares that there is no conflict of interests.


P<0.0001) and non-adherence  to adjuvant HT were significantly associated with increased mortality. Interruption to tamoxifen in younger patients and in patients receiving surgery (OP) with adjuvant chemotherapy (CT) was associated with increasing mortality rate when compared with their counterparts. Non-adherence to AIs in both younger and senior age groups and in OP with CT group also resulted in increasing risk. Treatment interruption and non-adherence to adjuvant HT were found to be associated with the increasing all-cause mortality of the Asian BC women; a greater impact of interruption and non-adherence on mortality was especially found in the younger BC population.This study aimed to evaluate the survival rate of women with breast cancer (BC) comparing persistence versus interruption and adherence versus non-adherence to adjuvant hormonal therapy (HT) in Asian population. Newly-diagnosed BC women from 2003 to 2010 were retrospectively identified from the Taiwan National Health Insurance Research Database. HT prescriptions were extracted to define treatment interruption and medication possession ratio. Their impacts on mortality were estimated by Cox regression with time dependent covariates. Interruption (HR: 1.32; 95% CI: 1.20, 1.46;  Breast cancer (BC) is the most common cancer and leading cause of cancer-related deaths for women worldwide Non-adherence and non-persistence to HT is a complex and multifaceted issue, and currently there is no gold standard of measures; even the conventional computation and cut-off threshold of medication possession ratio (MPR) and definition of gap period  varied between studies Current evidence on the impacts of non-adherence and non-persistence to HT on BC mortality has predominately emerged from Western countries In Taiwan, BC is the most common cancer and the fourth leading cause of cancer death for women. The incidence of BC increased from 40 to 56.1 per 100,000 women from 1999 to 2008, and the median age of diagnosis was 51 years The Taiwan Health Insurance Research Database (NHIRD) contains all medical claims from 1995; this longitudinal, population-based dataset serves as an appropriate resource for studying long-term HT utilization and BC treatment outcomes. Therefore, this study aimed to evaluate the impact of interruption and non-adherence to long-term adjuvant HT on survival outcome of BC women in Taiwan.This retrospective cohort study was conducted from 2003 to 2011 using the Taiwan NHIRD after being granted ethics approval from the Institutional Review Board (IRB) of Kaohsiung Medical University Hospital (KMUH-IRB-20120047). Informed consent was waived by the IRB. Taiwan NHI is a government-run, single-payer health insurance scheme established in 1995. It covered 97.1% of the entire population of 21.86 million in 2000 and 99.6% of the entire population (23.74 million) in 2010, and provides comprehensive benefit packages for a wide range of services The study cohort included adult (age ≥18 years old) women with newly-diagnosed BC from 2003 to 2010, without other concomitant cancers, who underwent surgical operations (breast-conserving surgery or mastectomy) as initial treatment and received at least 12-month adjuvant HT.BC patients were identified by screening BC-related International Classification of Diseases Revision 9 codes (ICD-9 codes 174) from both the individuals’ claim data and the Registry for Catastrophic Illness (a patient file in NHIRD). The disease index date was defined as the first positive BC diagnosis date. Since the ICD-9CM code 174 may be recorded for the purpose of BC screening, therefore, a second BC diagnosis recorded within 30 days would likely indicate a positive BC diagnosis, and in this case the first diagnosis date was defined as the index date instead. In addition, patients who had ICD-9 codes for other cancers  or benign lesions (210–239) prior to the index date, and those who did not undergo surgery were excluded from this study.From all HT  prescribed to the study cohort, the ‘days of supply’ (dispensed days) for each prescription and the ‘prescription duration’ (duration between the first and the last prescriptions plus the last prescription’s days of supply) for each patient were calculated. Patients whose prescription duration shorter than 12 months were excluded from the study sample since their prognosis may not directly be related to the interruption and non-adherence of medication. A sub-group analysis among patients whose duration of HT was less than 12 months (data not shown in tables) indicated that these excluded patients had less follow-up time (3.5±2.3 years), higher proportion of receiving target therapy (10.9%), and received AI only (19.4%) as compared to the study sample  of the mFrom individual patient’s HT prescriptions issued during the study period, MPR to HT was derived from dividing patient’s ‘total days of supply’ (sum of days of supply from HT prescriptions) by ‘prescription duration’. A conventional cut-off point of MPR less than 80% was used to define ‘non-adherence’ Any gap period between two consecutive HT prescriptions for more than 180 days was defined as ‘interruption’ (i.e. non-persistence) Patients’ adjuvant HT utilization patterns for the two HT types (tamoxifen or AIs) were also categorized into five groups, including tamoxifen only, tamoxifen switched to AIs, AIs only, AIs switched to tamoxifen, and multiple switches between tamoxifen and AIs. Switching of HT was defined as when patient received an alternative type for more than three refills (at least 84–90 days).The mortality and date of death were identified from the Registry for Catastrophic Illness. Follow-up time was calculated from the disease index date to the date of death or to the end of study (31 December 2011) for censored patients.Adjusted covariates included age of diagnosis, income groups, Charlson Comorbidity Index (CCI) score, initial treatment strategies, HT initiated year, HT prescription duration. Patients’ age was categorized in four ranks . The three most recently updated NHI insured income ranks were used as a synonym for individual’s monthly income status. Individual’s concomitant conditions recorded within 12 months prior to index date were identified by screening the ICD-9 codes related to CCI, and then converted into a CCI score Individual’s BC treatment strategies, including primary surgery (OP), adjuvant chemotherapy (CT), radiation therapy (RT), HT and targeted therapy (TT) were identified by corresponding medical-order and drug codes; and those recorded within 12 months posterior to index date were defined as the initial treatment strategies. Patients were stratified by whether they received adjuvant CT into two groups, i.e. OP with or without CT. Trastuzumab was the only TT reimbursed by NHI from April 2002 for HER2 positive BC in accordance with a set of stringent criteria, and thus TT was not included as an adjusting covariate due to limited prescription data.The survival rate and all-cause mortality rate were compared between persistence and interruption, and between adherence and non-adherence groups. Overall survival rate was evaluated using Kaplan-Meier survival analysis and stratified by whether patients received initial adjuvant CT. The association between adjusted covariates (and the corresponding time-dependent covariates) and all-cause mortality were evaluated by both univariate and multivariable analyses using the Cox proportional hazards model, and the results were presented in hazard ratios (HR) and 95% confidence intervals (CIs).The interruption- and non-adherence-associated HRs were also evaluated according to age (<50 or ≥50 years) and initial treatment (OP with or without CT) subgroups, and stratified in the four HT utilization patterns. Sensitivity analysis was conducted to assess the hazard ratios using various number of gaps or time to first gap  to define interruption, and various MPR cut off levels  to define non-adherence. All analyses were conducted by using SAS version 9.2 .During the 8-year inclusion period, 58,601 adult women with newly-diagnosed BC were identified, accounting for 99.4% BC cases reported to the Taiwan Cancer Registry in the same period. Therefore, we identified the representative newly-diagnosed BC women in this study. Overall, 30,573 patients fulfilling the study criteria were included; with a mean diagnosed age of 52.1±11.6 years, of whom 47% were diagnosed at the age of less than 50 years, and the majority (70%) of the patients had no apparent co-morbidity (CCI scored 0) .P<0.0001), with higher proportions of patients without any comorbidity .The total follow-up time was 144,438.8 patient-years (mean: 4.7±2.1 years per patient), and the mean HT prescription duration was 3.6±1.6 years. Of the five HT utilization patterns, most patients (65.9%) received tamoxifen only. Comparing the two initial treatment groups, patients who received adjuvant CT were significantly younger  were younger than those in the persistence  group (mean age: 50.6±11.3 vs. 52.4±11.6 years). The interruption group also had higher proportions of patients undergoing mastectomy (73.8% vs. 68.8%), receiving CT (74.7% vs. 68.4%) and TT (13.2% vs. 3.4%); and a lower proportion receiving tamoxifen only (49.2% vs. 68.9%), as opposed to other HT utilization patterns (p<0.0001) (Patients in the interruption group (<0.0001) .P<0.0001)  against the adherence  groups. The non-adherence group had a significantly higher proportion receiving tamoxifen only (69.4% vs. 64.9%), as opposed to having other HT utilization patterns (<0.0001) .P<0.0001) (P = 0.0218). However, the differences of survival rates between interruption and persistence groups were smaller in patients without receiving CT than those receiving CT  , A. Simiiving CT , B. Simiiving CT , C and DP<0.0001) and non-adherence  were significantly associated with increased mortality risk. In addition, higher age ranks, lower income ranks, CCI score≥2, and receiving CT were also significantly associated with an increased mortality risk.In the multivariable analyses , interruThe interruption and non-adherence associated mortality risk was further investigated in subgroups according to age, treatment strategy and HT utilization patterns . For intP = 0.0227) to 90%  . The first interruption occurred after the first  or the second  year of initiating HT was significantly associated with increased mortality risk. In terms of non-adherence, different MPR cut-off levels were associated with increased mortality risk from 50%  .N = 30,573) who underwent OP and over 12-month adjuvant HT was included. Compared with reports from Western countries This population-based study, to our knowledge, is the first to evaluate the impacts of HT interruption and non-adherence on the long-term BC survival using claim-based database in Asia. A study population  and Makubate et al (2013), who reported an increase in mortality rates by 49% and 20% with non-adherence  Based on the conventional measures Interruption and non-adherence measures vary by different definitions and follow-up duration. A systematic review reported a wide range of prevalence of adherence (41∼72%) and non-persistence (31∼73%) to HT measured at the end of 5 years of treatment After adjusting different covariates, this study found elderly age, higher CCI score, lower income, receiving OP and CT , and receiving RT were influencing factors to the interruption- and non-adherence-related mortality. Of which, initial treatment strategies and HT utilization patterns may influence on interruption- and non-adherence-related mortality; hence the study cohort was stratified to avoid indication bias.In line with the National Comprehensive Cancer Network guidelines Patients who had CT had greater interruption- and non-adherence-related detrimental effect on survival rates than those who did not have CT . This imPrevious studies on evaluating the HT discontinuation- or non-adherence-related all-cause mortality were conducted on cohorts with mostly postmenopausal women and with stratification according to HT utilization patterns. Non-adherence to AIs may have a greater detrimental effect on survival because AIs have a shorter half-life (24 hours to 50 hours). In contrast, with a longer half-life , non-adherence to tamoxifen, such as delayed or missed doses may not jeopardize the benefit of tamoxifen ‘Early interruption’ of HT is a concern for BC treatment as the peak recurrence rate has been found to be within the first two years after surgery We acknowledge several limitations of using a claim-based dataset for medicine and a lack of information on out-of-pocket drug utilization and disease status. Although the out-of-pocket medical utilization is not recorded, as BC is categorized as a ‘catastrophic’ disease and a comprehensive and continuous coverage is provided by the NHI, it is unlikely that BC patients would pay out-of-pocket for the relatively costly, long-term medications (such as AIs). Information on disease status ) were limited, and to reduce the effect of other prognostic factors on our investigation of interruption and non-adherence on mortality, we conducted our analyses within the strata of treatment modality to ensure relatively homogeneous clinical and pathological conditions. In addition, menopausal status was also limited, and hence surrogate indicator, such as cut-off point at median menopausal age of 50 years, was used.To use MPR as a proxy for adherence, assumptions were made that the dispensing claim data could represent the amount of HT consumed by patients, and methodology commonly used in studies to measure prescription refill adherence in claim-based data Using a predominantly ethnic Chinese women population-based database, we found that interruption and non-adherence to adjuvant HT were independently associated with increase in all-cause mortality. Future research on exploring the reasons for HT interruption and non-adherence is needed in order to develop efficient interventions to ensure completion of recommended course and to gain the best benefit of HT in BC treatment.


YKL-40 is involved in inflammation and endothelial dysfunction, and is increased in patients with type 1 diabetes, with an independent association between increasing YKL-40 levels and increasing levels of albuminuria. YKL-40 is associated with atherosclerosis and an increased cardiovascular mortality in the general population. In the present study YKL-40 levels were examined in patients with type 2 diabetes (T2D) with increasing levels of albuminuria, known to be associated with an increased risk of cardiovascular disease.One-hundred-five patients with T2D were examined: 49 with normoalbuminuria , 35 with persistent microalbuminuria  and 21 with persistent macroalbuminuria/diabetic nephropathy . The control group consisted of 20 healthy individuals (C). Groups were matched according to age, gender and known duration of diabetes.Median levels (interquartile range) of serum YKL-40 were significantly higher in N and MA vs. C (86 (55-137) ng/ml and 84 (71-147) ng/ml, respectively vs. 41 (33-55) ng/ml, p < 0.01) and even higher in patients with DN (120 (83-220) ng/ml, p < 0.001 for all comparisons). YKL-40 levels correlated with urinary albumin/creatinine-ratio in the total group of participants . Significant intercorrelations of YKL-40 were found with age, duration of diabetes, systolic blood pressure, lipid levels, HbA1c and HOMA-IR. After adjustment for significant covariates, albuminuria was significantly associated with YKL-40 levels .YKL-40 levels are elevated in patients with T2D with an independent association between increasing YKL-40 levels and increasing levels of albuminuria. The study suggests a role of YKL-40 in the progressing vascular complications in patients with T2D. Albuminuria is a well-established independent predictor for diabetic nephropathy and is also a strong predictor for cardiovascular morbidity and mortality in both patients with type 1 (T1D) and type 2 diabetes (T2D) . The exaYKL-40 is a glycoprotein involved in inflammation and endothelial dysfunction. It is a growth factor for various cell types and has an important role in extracellular matrix remodeling and angiogenesis ,8. In thThe objective of the present study was to evaluate serum YKL-40 in patients with T2D and with increasing levels of albuminuria.One-hundred-five patients with T2D were examined: 49 with normoalbuminuria , 35 with persistent microalbuminuria  and 21 with persistent macroalbuminuria/diabetic nephropathy . The control group consisted of 20 healthy individuals (C). Groups were matched according to age, gender and known duration of diabetes.http://mdrd.com/. Insulin resistance was estimated using the HOMA model http://www.dtu.ox.ac.uk/homacalculator/index.php. Urinary albumin was measured by immunoturbidimetry on a Cobas Bioanalyzer  from early morning spot urine collections. Lower detection limit was 1 mg/l, coefficients variations < 4.0%. Urinary creatinine was measured by a Jaffé reaction rate, using a kinetic principle to eliminate pseudocreatinines. Urinary albumin excretion was determined as UACR. The degree of albuminuria was confirmed by at least 2 consecutive tests. Diabetic retinopathy was assessed in most patients by fundus photography after pupillary dilatation and graded as nil, simplex or proliferative retinopathy.Plasma samples were taken as part of the first routine visit (inclusion) in the morning after an overnight fast and were stored at - 80°C until analysis. Beside routine analyses, analyses of serum YKL-40 were measured using ELISA method . Measuring range was 20-300 ng/ml, with intra- and interassay coefficients of variation of 5.8% and 6.0%. Glomerular filtration rate (GFR) was estimated using the 4 variable Modification of Diet in Renal Disease GFR formula  The study was approved by the Danish Data Protection Agency (id. 30122009.HEH.O.JF) and the local ethics committee of Copenhagen (KA 03065) and investigations conformed to the principles of The Helsinki Declaration.Following a test of statistical normality, data values are presented as mean ± SD or as median and interquartile range (IQR). For continuous variables, comparisons between the group of patients with T2D and the group of control subjects were performed with One-Way ANOVA. Mann-Whitney test was used if Levene's test for equality of variance was significant, or if a variable exhibited a clear non-Gaussian distribution. If data had a non-Gaussian distribution data were logarithmically transformed. Analyses of intercorrelations and associations were performed using univariate and multivariate linear regression analysis. The χ2-test was used for categorical variables.P values were two-sided, and p < 0.05 was considered statistically significant. All analyses were made with the statistical software package SPSS .Clinical data for the control group and the T2D patients with different levels of albuminuria is shown in Table Median YKL-40 levels were significantly different between groups with increasing YKL-40 levels with increasing levels of albuminuria (p < 0.001). We found no difference in eGFR between the different groups (p = 0.61). We did find a significant difference in UACR between the different groups (p < 0.001), even though most of the patients had UACR levels already reduced by antihypertensive medication, which was not stopped when spot urine samples were collected for the study. There was a significant difference in use of antihypertensive drugs between the groups (p < 0.001), with as expected more patients treated in the micro- and macroalbuminuric groups.We found no significant difference in systolic or diastolic blood pressure between the groups for all comparisons or comparisons of the diabetic groups. We did find significant differences in cholesterol and HbA1c levels (p < 0.001) between all groups due to differences between controls and the diabetic groups in total. No significant differences in cholesterol or HbA1c were found the diabetic groups in between.Simple and multiple regression analyses showed correlation of YKL-40 with UACR in the total group of participants  Table . SignifiIn a multiple regression model adjusting for the significant covariates , albuminuria was still found to be significantly associated with YKL-40 levels . Moreover, significant associations were also found with total cholesterol  and LDL-cholesterol .UACR was significantly associated with YKL-40, BMI, HbA1c and systolic blood pressure (0.001 < p < 0.02). A multiple regression analyses adjusted for significant covariates demonstrated that YKL-40 was associated with albuminuria .More patients with T2D were treated with statins, when compared to the control subjects (p < 0.001), but there were no differences in the number of T2D treated between the groups. No differences were seen in levels of YKL-40 in the whole group of T2D patient treated with statins when compared to non-treated patients (p = 0.18), nor in the individual groups (0.1 < p < 0.9).2 = 8.6, p = 0.036). Patients with retinopathy had significantly higher levels of YKL-40 (p = 0.01), only due to differences between normoalbuminuric and microalbuminuric patients (p = 0.021). More patients with albuminuria had retinopathy .At baseline, only a limited number of subjects had a history of myocardial infarction, stroke or symptoms of intermittent claudicatio. No differences in YKL-40 levels were found in patients with a history of CVD (any), when compared to patients without these macrovascular complications (p = 0.47). However, more patients with macroalbuminuria had CVD  mortality rate in an elderly part of the general population without diabetes and CVD, after adjustment for known CVD risk factors and markers . YKL-40 It has been demonstrated that statins, through their capacity to inhibit inflammation, can reduce the increased inflammatory activities seen with progression of atherosclerosis, and this is correlated with the reduction in calcification . In the Vascular endothelial dysfunction is an important factor in the pathogenesis of diabetic micro- and maroangiopathy . YKL-40 YKL-40 also seems to promote tumor angiogenesis in colon and breast cancer , an effeThere are limitations of the present study. We have performed a cross-sectional analysis of YKL-40 levels in relation to albuminuria in patients with T2D; however long-time studies are needed to clarify whether an increase in YKL-40 levels in both T1D and T2D patients is a consequence of a general progressing atherosclerotic disease, or whether YKL-40 can predict or contribute to microvascular and macrovascular disease in these patients. Moreover, in the present study no other markers of endothelial dysfunction and inflammation were investigated, e.g. high-sensitive C-reactive protein (hsCRP). But in contrast to most inflammatory markers, YKL-40 is locally produced at the site of inflammation ,24 and pIn conclusion, YKL-40 levels are elevated in patients with T2D with an independent association between increasing YKL-40 levels and increasing levels of albuminuria after adjustment for UACR, age and other significant covariates. A role of YKL-40 in the gradually progressing vascular complications in patients with diabetes is suggested, with YKL-40 being a possible early marker of the widespread atherosclerotic complications in diabetic patients.The authors declare that they have no competing interests.AKR and EO participated in the design, coordination of the study, and in analysis and interpretation of data and drafted the manuscript. HV conceived of the study and participated in the design, coordination, conduct of the study, performed the statistical analysis and helped to draft the manuscript. All authors have read and approved the final version of the manuscriptNone of the authors declare any financial disclosures.


US visceral and subcutaneous-abdominal depths correlated with MRI quantified IA-AT  and SCA-AT  volumes, respectively. In CBGS, mean US-visceral depths increased by ~20% between ages 3 and 12 months (P < 0.0001) and at both ages were lower in infants breast-fed at 3 months than in other infants. US-visceral depths at both 3 and 12 months were inversely related to skinfold thickness at birth . In contrast, US-subcutaneous-abdominal depth at 3 months was positively related to skinfold thickness at birth (P = 0.004). US measures can rank infants with higher or lower IA-AT and SCA-AT. Contrasting patterns of association with visceral and subcutaneous-abdominal adiposities indicate that they may be differentially regulated in infancy.Other imaging techniques to quantify internal-abdominal adiposity (IA-AT) and subcutaneous-abdominal adiposity (SCA-AT) are frequently impractical in infants. The aim of this study was twofold: (a) to validate ultrasound (US) visceral and subcutaneous-abdominal depths in assessing IA-AT and SCA-AT from MRI as the reference method in infants and (b) to analyze the association between US abdominal adiposity and anthropometric measures at ages 3 months and 12 months. Twenty-two infants underwent MRI and US measures of abdominal adiposity. Abdominal US parameters and anthropometric variables were assessed in the Cambridge Baby Growth Study (CBGS),  Childhood obesity has become a major public health issue and its prevalence is increasing worldwide –3. More Several epidemiological studies have reported that early life factors, such as impaired fetal growth or excess postnatal weight gain, are associated with later obesity and related comorbidities –15. GrowUltrasound (US) has been assessed as a noninvasive estimate of IA-AT and SCA-AT. US-visceral depth and US abdominal-subcutaneous depth have been shown to be reliable and reproducible estimates of IA-AT and SCA-AT, respectively, when compared to CT or MRI in adults and in adolescents –24. HoweThe validation study was carried out in a convenience sample of 22 healthy term singleton newborn infants (10 boys and 12 girls). Mothers and babies were recruited from the Neonatal Unit and postnatal wards of the Chelsea and Westminster Hospital, London, UK, between 2008 and 2009 and attended the Robert steiner MRI Unit, Hammersmith Hospital, London, UK. This study was approved by the Hammersmith and Queen Charlotte's & Chelsea Hospital research ethics committee. Written parental consent was obtained prior to the participants' visit.Details of the study have been described elsewhere . BrieflyIn the validation study, weight, length, and waist circumference (WC) were measured by one of three trained clinical research fellows. Weight was measured using a Marsden Professional Baby Scale  and recorded to the nearest 0.1 kg. Crown-heel length was measured with a Rollameter, a recumbent infant board with a sliding footboard . WC was measured at the midpoint between the inferior border of the costal margin and the anterior superior iliac crests using a D-loop tape measure  . 3. SD scores (SDS) were derived for weight and length by comparison to the 1990 British reference [minus cohort mean)/cohort SD], and then the overall skinfold thickness SDS was calculated as the mean of the four skinfold SD scores in each individual. The relative intraobserver technical error of measurement (TEM) for length ranged between 0.03% and 0.05%, for quadriceps ranged between 0.4% and 0.6%, for triceps ranged between 1.9% and 2.4%, for subscapular ranged between 1.7% and 2.8% and for flank ranged between 0.8% and 2.0%. The relative interobserver TEM was 0.7% for length, 2.0% for quadriceps, 2.9% for subscapular, 2.2% for triceps, 3.2% for flank. The calculations were based on repeated measurements in 12 infants.In CBGS, infants were measured at birth, 3 months, and 12 months by trained paediatric nurses or research assistants. Weight was measured to the nearest 1 g using a SECA 757 digital scale (Chasmors Ltd.) and length using a Kiddimeter (Chasmors Ltd). WC was measured as described previously. Triceps, quadriceps, flank, and subscapular skinfold thicknesses were measured in triplicate on the left side of the body using Holtain calipers (Chasmors Ltd). The triceps skinfold was measured halfway between the acromial process and the olecranon. The quadriceps skinfold was taken from a vertical line over the quadriceps muscle at midline of the thigh, and half way between the top of the patella and the inguinal crease. The flank (posterior suprailiac) skinfold was taken from the diagonal plane in line with the natural angle of the iliac crest taken in the posterior axillary line immediately posterior to the iliac crest. The skinfold was taken at the oblique angle below the left scapula . Ponderaeference . SeparatUS-visceral depth and US-subcutaneous-abdominal depth were measured using a Logiq Book XP ultrasound, with a 3C MHZ -RS abdominal curved array transducer . For both measures, the transducer was positioned where the xiphoid line intercepted the WC measurement plane, and the images were taken during expiration. US-visceral depth was measured on a longitudinal plane with a probe depth of 9 cm and was defined as the distance between the peritoneal boundary and the corpus of the lumbar vertebra. US-subcutaneous abdominal depth was measured at the same location, but on a transverse plane with a probe depth of 4 cm, and was defined as the distance between the cutaneous boundary and the linea alba. The image was captured when the transducer just had contact with the skin to avoid compressing the subcutaneous adipose area. In the validation study, the US measures were performed by one of two trained operators and in CBGS by one of four trained operators. The relative intraobserver technical error of measurement (TEM) ranged between 0.3% and 1.7% for US-visceral depth, and 1.1% and 2.6% for US-subcutaneous-abdominal depth, and the relative interobserver TEM was 3.2% for US-visceral depth 3.6% for US-subcutaneous-abdominal depth, based on repeated measurements in 12 infants. In the validation study, qualitative information on the feasibility and acceptability of US was collected from the participants using open-ended questions. The MRI procedure used in the validation study is described elsewhere . Brieflyt-tests. For validation purposes, Pearson's correlation coefficients were used to describe the associations between IA-AT or SCA-AT and the US and anthropometric variables. Multiple regression was used to test the added contribution of US depths to anthropometry in explaining the variance in IA-AT or SCA-AT including the root mean square error (RMSE). Statistical analyses were performed using STATA version 11.0 (StataCorp Ltd.). Means and standard deviations are presented separately for boys and girls and sex differences were tested using unpaired For CBGS, Pearson's correlation coefficients were used to describe cross-sectional associations between US depths at 3 or 12 months and anthropometric variables. Associations between growth parameters at birth (birth weight and skinfolds SDS) and US depths at 3 or 12 months were tested using linear regression models. Associations were similar in both sexes, so all analyses were performed in the total sample with adjustment for sex. Further adjustment for current size (weight or skinfolds SDS) was included in the final models. Colinearity between parameters in the same model was quantified using the variance inflation factor (VIF); models with VIF > 5 were considered invalid . To explP < 0.05.All body composition variables and the residuals of the regression models were normally distributed. Statistical significance was set at 3, SCA-AT 104 (59–202) cm3; US-visceral depth 2.0 (1.2–3.0) cm; and US-subcutaneous abdominal depth 0.30 (0.2–0.4) cm. In the 22 newborn infants, mean range for age was 10.6 (6–19) days; gestational age at birth 39.9 (37.1–40.8) weeks; weight 3.3 (2.5–3.9) kg; length 53.1 (47–57) cm; WC 34 (29–39) cm; IA-AT 18 (8–32) cmr = 0.48; P = 0.02) and US-subcutaneous abdominal depth , and these were higher than with any anthropometric variable , followed by weight . US-subcutaneous abdominal depth was also strongly positively correlated with total SC-AT , weight , and length . Examination of scatter plots . For the prediction of SCA-AT, the addition of US-subcutaneous abdominal depth to weight, sex and age improved the explained variance from 44% to 65% (P = 0.1). Accordingly, addition of the US parameters substantially reduced the root mean square error (RMSE) terms for SCAT-AT for IA-AT  or 12 months (N = 495) are summarised in P < 0.0001), despite no difference in gestational age (P > 0.05). Boys remained heavier and taller than girls at 3 and 12 months, and boys had slightly greater mean US-visceral depth than girls at 12 months (P = 0.04) but not at 3 months (P = 0.9). Characteristics of CBGS infants with US measures at age 3 months (P < 0.0001). In this longitudinal sample US-visceral depth showed only weak tracking between 3 and 12 months . In contrast the inter-correlation coefficients between 3–12 months were stronger for mean skinfold thickness SDS , ponderal index , US-subcutaneous abdominal depth , WC , weight , and length . Despite these marked changes during infancy, US-visceral depths were consistently lower at both 3 and 12 months in infants who were exclusively breast-fed at age 3 months compared to other infants . US-visceral depth was unrelated to time from last feed at 3 months  and 12 months .Mean US-visceral depth at age 12 months was 22% higher in boys and 17% higher in girls at 12 months than at 3 months. In contrast, mean US-subcutaneous abdominal depth and skinfold thickness did not change with age. The apparent increase in US-visceral depth was confirmed in the 360 infants with repeat measures at both 3 and 12 months  and with mean skinfold thickness SDS at 12 months (P = 0.02). In contrast, US-subcutaneous abdominal depth at both 3 and 12 months was positively associated with all measures of current body size (P < 0.005). In cross-sectional analyses , US-viscP = 0.06) and 12 months (P = 0.03) showed inverse trends or associations with skinfold thickness at birth, and these inverse associations strengthened on adjustment for current skinfold thickness  (positively associated with skinfold thickness at birth (P = 0.004), but not at age 12 months (P = 0.1) and no associations remained on adjustment for current skinfolds (P = 0.01 at both 3 and 12 months).In models without adjustment for current body size, US-visceral depth at 3 months (= 0.009) . In contkinfolds . In unadOur validation study results showed that US abdominal depth provides acceptable accuracy in estimating IA-AT and SCA-AT volumes assessed by MRI in infants. The US measures showed stronger correlations with IA-AT and SCA-AT than did the traditional anthropometric variables, and the addition of US measures to those variables substantially improved the predictions of IA-AT and SCA-AT. The precision of our models was significantly improved as RMSE for IA-AT and SCA-AT substantially decreased. Furthermore, the reproducibility and reliability of the US measures were high as indicated by low inter- and intraobserver technical errors of measurement. In addition, the ultrasound method was highly acceptable to parents as it was faster to perform than MRI and no separation from their infants was required. By contrast, the actual MRI scanning time is approximately 12 minutes, but the whole procedure including preparation time to settle the infant can take up to one hour.We acknowledge that our validation study has some limitations. In particular, it was performed in newborns at age range 6–19 days, rather than at 3 or 12 months as in CBGS. This is because the reference imaging techniques, MRI and CT, are not feasible for research studies at those later ages, as discussed previously. However, our findings are consistent with positive reports in adults and adolescents comparing abdominal US to MRI , 31, 32.n = 22). In fact this study had 80% power to detect a Pearson's correlation coefficient higher than 0.56 with a type I error of 5%. Our inclusion criteria were limited to only healthy newborns (birth weight range 2.5–3.9 kg) due to the need to travel to a research site some miles from their place of birth. We anticipate that the inclusion of infants with more extremes of underweight/thinness and macrosomia would increase the strength of the observed correlations. We were unable to test absolute validity using the Bland-Altman analysis because this method requires the different measurements to be reported in the same units in order to calculate the degree of bias on the raw measurement scale. In addition, no existing prediction equations were available for IA-AT and SC-AT from US measures based on US measures in this age group. Future independent studies should test the absolute validity of the prediction models derived in this study. However, our main purpose was not to develop prediction models, but rather to analyze the associations between anthropometric variables, age, and gender with US parameters.Secondly, the sample size in our validation study was small . Indeed, US-subcutaneous abdominal depth showed a slightly stronger correlation with IA-AT , but was more strongly related to SCA-AT and hence US-visceral depth was the more specific marker of IA-AT. In contrast, the correlations between US-visceral depth and IA-AT were 0.80–0.82 in older adults and 0.64–0.72 in adolescents [Finally, the correlation between US-visceral depth and IA-AT was only moderate (lescents –24. Lowelescents , such colescents , 39, andlescents . Therefolower subcutaneous abdominal depth at age 3 months, but greater visceral depths at ages 3 and 12 months, suggesting a differential regulation of these adipose tissue compartments. The stronger visceral depth associations that we observed with lower skinfold thickness at birth rather than lower birth weight suggest that these birth measures may be proxies for fetal growth restraint during the later antenatal period. In support of this notion, our previous studies using MRI in newborns reported that growth-restricted and extremely preterm infants have reduced SCAT but preserved IA-AT mass [In the CBGS cohort study, we found that infants with lower skinfold thickness at birth tended to have -AT mass , 28. OurWe also observed that the associations between skinfold thickness at birth and infancy visceral depth strengthened with further adjustment for current skinfold thickness. Some investigators have argued that adjustment for current size could potentially introduce bias due to overcontrolling . HoweverTherefore, postnatal factors related to infancy gains in skinfold thickness may influence the accumulation of visceral adipose tissue at 3 and 12 months. Our observation of weak tracking in visceral depth indicates wide between-individuals variation in the rate of accumulation of visceral adipose tissue during infancy, although measurement error and imprecision are likely contributing factors to this estimate. Our observed associations with breastfeeding indicate that postnatal nutrition may influence the accumulation of visceral adipose tissue in infancy.In conclusion, US abdominal depths were better than anthropometric measures in ranking infants with higher or lower IA-AT and SCA-AT volumes and may be applicable to large epidemiological studies at young ages when MRI and CT imaging techniques are infeasible. Application of these US measures in a large birth cohort study showed that visceral and subcutaneous-abdominal depths differed in their changes with age and in their patterns of association with antenatal and postnatal factors, suggesting that IA-AT and SCA-AT may be differentially regulated in the first year of life.